Line 138: | Line 138: | ||
<p>Using SEAP<sup>KDEL</sup> we were able to confirm that the reporter is not present in the cell medium without coexpression of erTEVp. When erTEVp was cotransfected with | <p>Using SEAP<sup>KDEL</sup> we were able to confirm that the reporter is not present in the cell medium without coexpression of erTEVp. When erTEVp was cotransfected with | ||
the reporter, we detected a large increase in enzymatic activity in the medium (<ref>2</ref>). | the reporter, we detected a large increase in enzymatic activity in the medium (<ref>2</ref>). | ||
− | <div | + | <div style = "clear:left; float:left"> |
<figure data-ref="2"> | <figure data-ref="2"> | ||
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/6/61/T--Slovenia--6.2.2.png" > | <img class="ui medium image" src="https://static.igem.org/mediawiki/2016/6/61/T--Slovenia--6.2.2.png" > |
Revision as of 15:33, 15 October 2016
Protease-based inducible secretion
Fast sensing and fast signaling pathway were developed using protease-based pathway. In the final step for the construction of rapidly responding cells we wanted to implement a fast output that would not require a slow transcription/translation biosynthesis of new proteins. We decided to engineer a system capable of regulated secretion of a protein using genetically encoded components.
To achieve a fast regulated cellular response resulting in the release of a protein, we decided to mimic the release of insulin from beta cells where the protein of interest is pre-formed and present in the cell in secretory granules. In contrast to the specialized storage and release mechanism of insulin from beta cells we wanted to develop a more general and modular solution by making use of components already existing in different types of cells. Additionally, there should be minimal leakage from the protein depot in the uninduced state and after induction secretion from the cell should be fast.
Many proteins that reside on the membrane or in the lumen of the ER contain short peptide signals. Proteins present in the lumen of the ER contain a KDEL C-terminal sequence
(Lys-Asp-Glu-Leu) while type I transmembrane (TM) proteins contain a dilysine (KKXX) motif on their C-terminus (cytosolic side).
Our idea was that if we proteolytically remove the retention signal, the protein of interest would no longer be retrieved back to the ER and could be secreted from the cell. To achieve this we designed two types of secretory reporters, one type based on the luminal retention using KDEL sequence and the other based on the transmembrane retention with a KKMP sequence. In each case, the retention sequence was preceded by a TEVp cleavage site to allow for inducible secretion, which could be replaced by any other peptide target of our orthogonal protease set.
Secretion from the ER lumen
To achieve and detect the inducible secretion from the ER lumen, we created two reporter constructs with a cleavable KDEL sequence targeted to the ER lumen: SEAPKDEL and TagRFPKDEL. Those proteins contained a protease target motif between the reporter domain and the KDEL domain, aimed to enable protein secretion after the proteolytic cleavage. We used a TEVp variant (erTEVp) for all of our experiments with luminal retention.
Results
When the TagRFPKDEL reporter (1A) was expressed in the ER without an active erTEVp we confirmed its localization in the ER with confocal microscopy (1B). Additionally, we could not detect any TagRFP in the cell medium with Western blotting. When erTEVp was present and active in the ER, the KDEL sequence was removed from the reporter and the protein was secreted from the cell, which we detected with Western blot (1C), demonstrating that proteolytic activity in the ER can regulate protein secretion.
![](https://static.igem.org/mediawiki/2016/9/9e/T--Slovenia--6.2.1.png)
(A) Scheme of the reporter with cleavable KDEL retention signal and protease target motif. (B) The reporter with the KDEL retention signal was localized in the ER. HEK293T cells were transfected with the indicated reporters and in (C) also with erTEVp. Localization was detected with confocal microscopy. (C)The reporter was detected in the medium of cells only when cotransfected with erTEVp. HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium.
Using SEAPKDEL we were able to confirm that the reporter is not present in the cell medium without coexpression of erTEVp. When erTEVp was cotransfected with the reporter, we detected a large increase in enzymatic activity in the medium (2).
![](https://static.igem.org/mediawiki/2016/6/61/T--Slovenia--6.2.2.png)
HEK293T cells were transfected with indicated reporter and erTEVp. Increased SEAP activity was detected in the medium of cells expressing both reporter and erTEVp protease.